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Alternative splicing of CD200 is regulated by an exonic splicing enhancer and SF2/ASF.

Chen Z, Ma X, Zhang J, Hu J, Gorczynski RM - Nucleic Acids Res. (2010)

Bottom Line: A truncated CD200 (CD200(tr)) resulting from alternative splicing has been identified and characterized as a functional antagonist to full-length CD200.Deletion or mutation of the ESE site decreased expression of the full-length CD200.Knockdown of expression of SF2/ASF resulted in the same splicing pattern as seen after deletion or mutation of the ESE, whereas overexpression of SF2/ASF increased expression of the full-length CD200.

View Article: PubMed Central - PubMed

Affiliation: Transplant Research Division, Department of Surgery and Immunology, The Toronto Hospital, University of Toronto, Toronto, Canada. zhiqi.chen@utoronto.ca

ABSTRACT
CD200, a type I membrane glycoprotein, plays an important role in prevention of inflammatory disorders, graft rejection, autoimmune diseases and spontaneous fetal loss. It also regulates tumor immunity. A truncated CD200 (CD200(tr)) resulting from alternative splicing has been identified and characterized as a functional antagonist to full-length CD200. Thus, it is important to explore the mechanism(s) controlling alternative splicing of CD200. In this study, we identified an exonic splicing enhancer (ESE) located in exon 2, which is a putative binding site for a splicing regulatory protein SF2/ASF. Deletion or mutation of the ESE site decreased expression of the full-length CD200. Direct binding of SF2/ASF to the ESE site was confirmed by RNA electrophoretic mobility shift assay (EMSA). Knockdown of expression of SF2/ASF resulted in the same splicing pattern as seen after deletion or mutation of the ESE, whereas overexpression of SF2/ASF increased expression of the full-length CD200. In vivo studies showed that viral infection reversed the alternative splicing pattern of CD200 with increased expression of SF2/ASF and the full-length CD200. Taken together, our data suggest for the first time that SF2/ASF regulates the function of CD200 by controlling CD200 alternative splicing, through direct binding to an ESE located in exon 2 of CD200.

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SF2/ASF determines exon 2 inclusion or exclusion in endogenously or exogenously expressed CD200. siRNA against SF2/ASF was used to ‘knock-down’ its expression. (A) Western blotting was employed in Daudi and SK-N cells after treated with 2.5 μg siRNA. (1,4) no siRNA; (2,5) 2.5 μg siRNA; (3,6) scramble siRNA. β-Actin was used as a loading control. Absolute quantitative real-time RT-PCR was performed in Daudi cells (B and D) and SK-N cells (C and E) before and after treated with 2.5 μg of SF2/ASF siRNA. Scramble siRNA was used as negative control. Broken lines reflect that endogenous or exogenous expression of full-length CD200 decreased after knockdown of SF2/ASF relative to the control group (no siRNA treatment) (P < 0.05). Continuous lines reflect endogenous or exogenous expression of truncated CD200, which are increased after knockdown of SF2/ASF relative to the control group (no siRNA treatment) (P < 0.01).
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Figure 6: SF2/ASF determines exon 2 inclusion or exclusion in endogenously or exogenously expressed CD200. siRNA against SF2/ASF was used to ‘knock-down’ its expression. (A) Western blotting was employed in Daudi and SK-N cells after treated with 2.5 μg siRNA. (1,4) no siRNA; (2,5) 2.5 μg siRNA; (3,6) scramble siRNA. β-Actin was used as a loading control. Absolute quantitative real-time RT-PCR was performed in Daudi cells (B and D) and SK-N cells (C and E) before and after treated with 2.5 μg of SF2/ASF siRNA. Scramble siRNA was used as negative control. Broken lines reflect that endogenous or exogenous expression of full-length CD200 decreased after knockdown of SF2/ASF relative to the control group (no siRNA treatment) (P < 0.05). Continuous lines reflect endogenous or exogenous expression of truncated CD200, which are increased after knockdown of SF2/ASF relative to the control group (no siRNA treatment) (P < 0.01).

Mentions: As previously described, the full-length CD200 was expressed predominantly in brain and neuronal cells. One explanation of this observation is that the expression of SF2/ASF is higher in neuronal cells and brain. To test this hypothesis, we assessed SF2/ASF levels in Daudi and SK-N cells by Western blotting. As shown in Figure 6A, the natural level of SF2/ASF was clearly higher in SK-N cells than in Daudi cells.Figure 6.


Alternative splicing of CD200 is regulated by an exonic splicing enhancer and SF2/ASF.

Chen Z, Ma X, Zhang J, Hu J, Gorczynski RM - Nucleic Acids Res. (2010)

SF2/ASF determines exon 2 inclusion or exclusion in endogenously or exogenously expressed CD200. siRNA against SF2/ASF was used to ‘knock-down’ its expression. (A) Western blotting was employed in Daudi and SK-N cells after treated with 2.5 μg siRNA. (1,4) no siRNA; (2,5) 2.5 μg siRNA; (3,6) scramble siRNA. β-Actin was used as a loading control. Absolute quantitative real-time RT-PCR was performed in Daudi cells (B and D) and SK-N cells (C and E) before and after treated with 2.5 μg of SF2/ASF siRNA. Scramble siRNA was used as negative control. Broken lines reflect that endogenous or exogenous expression of full-length CD200 decreased after knockdown of SF2/ASF relative to the control group (no siRNA treatment) (P < 0.05). Continuous lines reflect endogenous or exogenous expression of truncated CD200, which are increased after knockdown of SF2/ASF relative to the control group (no siRNA treatment) (P < 0.01).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
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Figure 6: SF2/ASF determines exon 2 inclusion or exclusion in endogenously or exogenously expressed CD200. siRNA against SF2/ASF was used to ‘knock-down’ its expression. (A) Western blotting was employed in Daudi and SK-N cells after treated with 2.5 μg siRNA. (1,4) no siRNA; (2,5) 2.5 μg siRNA; (3,6) scramble siRNA. β-Actin was used as a loading control. Absolute quantitative real-time RT-PCR was performed in Daudi cells (B and D) and SK-N cells (C and E) before and after treated with 2.5 μg of SF2/ASF siRNA. Scramble siRNA was used as negative control. Broken lines reflect that endogenous or exogenous expression of full-length CD200 decreased after knockdown of SF2/ASF relative to the control group (no siRNA treatment) (P < 0.05). Continuous lines reflect endogenous or exogenous expression of truncated CD200, which are increased after knockdown of SF2/ASF relative to the control group (no siRNA treatment) (P < 0.01).
Mentions: As previously described, the full-length CD200 was expressed predominantly in brain and neuronal cells. One explanation of this observation is that the expression of SF2/ASF is higher in neuronal cells and brain. To test this hypothesis, we assessed SF2/ASF levels in Daudi and SK-N cells by Western blotting. As shown in Figure 6A, the natural level of SF2/ASF was clearly higher in SK-N cells than in Daudi cells.Figure 6.

Bottom Line: A truncated CD200 (CD200(tr)) resulting from alternative splicing has been identified and characterized as a functional antagonist to full-length CD200.Deletion or mutation of the ESE site decreased expression of the full-length CD200.Knockdown of expression of SF2/ASF resulted in the same splicing pattern as seen after deletion or mutation of the ESE, whereas overexpression of SF2/ASF increased expression of the full-length CD200.

View Article: PubMed Central - PubMed

Affiliation: Transplant Research Division, Department of Surgery and Immunology, The Toronto Hospital, University of Toronto, Toronto, Canada. zhiqi.chen@utoronto.ca

ABSTRACT
CD200, a type I membrane glycoprotein, plays an important role in prevention of inflammatory disorders, graft rejection, autoimmune diseases and spontaneous fetal loss. It also regulates tumor immunity. A truncated CD200 (CD200(tr)) resulting from alternative splicing has been identified and characterized as a functional antagonist to full-length CD200. Thus, it is important to explore the mechanism(s) controlling alternative splicing of CD200. In this study, we identified an exonic splicing enhancer (ESE) located in exon 2, which is a putative binding site for a splicing regulatory protein SF2/ASF. Deletion or mutation of the ESE site decreased expression of the full-length CD200. Direct binding of SF2/ASF to the ESE site was confirmed by RNA electrophoretic mobility shift assay (EMSA). Knockdown of expression of SF2/ASF resulted in the same splicing pattern as seen after deletion or mutation of the ESE, whereas overexpression of SF2/ASF increased expression of the full-length CD200. In vivo studies showed that viral infection reversed the alternative splicing pattern of CD200 with increased expression of SF2/ASF and the full-length CD200. Taken together, our data suggest for the first time that SF2/ASF regulates the function of CD200 by controlling CD200 alternative splicing, through direct binding to an ESE located in exon 2 of CD200.

Show MeSH
Related in: MedlinePlus